PROJECT SUMMARY Patients with HIV infection are living longer thanks to combination antiretroviral therapy (cART), but they often necessitate treatment for comorbidities. Our long-term goal is to improve the lives of HIV-infected patients with comorbid cancer, a main cause of mortality in the cART era. HIV-infected patients with comorbid cancer have a higher risk of dying as a result of their cancer than non-HIV-infected patients. Several studies have demonstrated an association between ineffective (non-suppressive) cART and poor response to cancer chemotherapy (CHEMO) and mortality. Identification of the factors controlling the effectiveness of cART in the context of CHEMO could reduce cancer deaths in HIV-infected patients. We reasoned that CHEMO could modulate the antiviral activity of cART, based on the observation that inhibition of cellular thymidylate synthase (TS), a main target of CHEMO, alters intracellular concentrations of various nucleotides. Our goal is to evaluate the effects of TSi on the antiviral activity of cART. Our hypothesis is that TS inhibitors (TSi) can have inhibitory and enhancing effects on cART, impacting development of HIV resistance and the HIV reservoir in vivo. In Preliminary Studies, gemcitabine (GCB) enhanced the anti-HIV activities of NRTIs TFV, ABC and FTC in primary cells. In contrast, pemetrexed (PTX) inhibited FTC and 3TC activities. Mechanistic studies showed that PTX lowered the concentrations of FTCtp relative to its competing endogenous nucleotide (dCTP), which is a determinant of FTC efficacy in primary cells. Consistent with these data, the TFV/FTC/dolutegravir combination suppressed HIV in the absence, but not in the presence, of PTX. These data suggested that HIV-infected patients treated with certain cART/TSi combinations could actually have only 2, rather than 3, active ARTs, decreasing the overall potency of cART, increasing the risk of drug resistance and expanding HIV reservoirs. Preliminary Studies in humanized mice demonstrated that GCB enhances TFV inhibition of plasma HIV RNA by up to 6 log10 units, whereas PTX abrogated FTC activity. These data are the first evidence that TSi-based CHEMO can have opposing effects on cART efficacy in vivo, impacting control of HIV and thereby development of viral resistance and size of the reservoir. This proposal will evaluate the effects of PTX, GCB and other approved TSi on cART efficacy. There are three Specific Aims. Specific Aim 1: To evaluate and characterize the effects of TS inhibitors (TSi) on the anti-HIV activities of NRTIs in primary CD4+ T cells in vitro and in humanized mice. Specific Aim 2: To evaluate the impact of cART/TSi combinations on the resting CD4 T cell HIV reservoir. Specific Aim 3: To evaluate durability (lack of HIV resistance emergence) and toxicity of cART/TSi combinations during long-term treatment of HIV infection. Currently there are no treatment guidelines for the use of cART and CHEMO in HIV-infected patients with cancer, but this proposal could help to delineate guidelines and decrease cancer deaths in patients with HIV.